PDBsum entry: 1e40

Hydrolase

PDB id: 1e40

Contents

Protein chain

483 a.a. *

Ligands

TRS

GLC-GLC-GLC

Metals

_CA ×4

_NA

Waters ×539

* Residue conservation analysis

PDB id:

1e40

Name:

Hydrolase

Title:

Tris/maltotriose complex of chimaeric amylase from b. Amyloliquefaciens and b. Licheniformis at 2.2a

Structure:

Alpha-amylase. Chain: a. Engineered: yes. Other_details: chimaeric structure consisting of residues 1 - 300 of b. Amyloliquefaciens and residues 301 - 483 of b. Licheniformis in complex with tris and maltotriose

Source:

Bacillus amyloliquefaciens. Organism_taxid: 1390. Expressed in: bacillus amyloliquefaciens. Expression_system_taxid: 1390. Other_details: synthetic gene

Resolution:

2.20Å R-factor: 0.130 R-free: 0.210

Authors:

A.M.Brzozowski,D.M.Lawson,J.P.Turkenburg, H.Bisgaard-Frantzen,A.Svendsen,T.V.Borchert,Z.Dauter, K.S.Wilson,G.J.Davies

Key ref:

A.M.Brzozowski et al. (2000). Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes. Biochemistry, 39, 9099-9107. PubMed id: 10924103 DOI: 10.1021/bi0000317

Date:

27-Jun-00 Release date: 21-Jun-01

Protein chain

P00692  (AMY_BACAM) -  Alpha-amylase

Seq: 514 a.a.

Struc: 483 a.a.*

* PDB and UniProt seqs differ at 32 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.1 - Alpha-amylase.
• Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biological process: metabolic process (2 terms)
• Biochemical function: catalytic activity (8 terms)

DOI no: 10.1021/bi0000317

Biochemistry 39:9099-9107 (2000)

PubMed id: 10924103

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

A.M.Brzozowski, D.M.Lawson, J.P.Turkenburg, H.Bisgaard-Frantzen, A.Svendsen, T.V.Borchert, Z.Dauter, K.S.Wilson, G.J.Davies.

ABSTRACT

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.